XIAP-mediated degradation of IFT88 disrupts HSC cilia to stimulate HSC activation and liver fibrosis

Activation of hepatic stellate cells (HSCs) plays a critical role in liver fibrosis. However, the molecular basis for HSC activation remains poorly understood. Herein, we demonstrate that primary cilia are present on quiescent HSCs but exhibit a significant loss upon HSC activation which correlates with decreased levels of the ciliary protein intraflagellar transport 88 (IFT88). Ift88-knockout mice are more susceptible to chronic carbon tetrachloride-induced liver fibrosis. Mechanistic studies show that the X-linked inhibitor of apoptosis (XIAP) functions as an E3 ubiquitin ligase for IFT88. Transforming growth factor-β (TGF-β), a profibrotic factor, enhances XIAP-mediated ubiquitination of IFT88, promoting its proteasomal degradation. Blocking XIAP-mediated IFT88 degradation ablates TGF-β-induced HSC activation and liver fibrosis. These findings reveal a previously unrecognized role for ciliary homeostasis in regulating HSC activation and identify the XIAP–IFT88 axis as a potential therapeutic target for liver fibrosis.

(A-C) Immunofluorescence images of primary cilia on hepatocytes (A), BECs (B), and PVCs (C) from CCl 4 -treated mice (n = 10 mice).Scale bars, 10 μm.(D) Quantification of the percentage of ciliated cells in hepatocytes, PVCs, and BECs from mice described in (A-C) (n = 10 mice).To quantify the percentage of ciliated cells (D), >200 cells from six fields were analyzed for each mouse.The same group of mice (n = 10 mice for each time point) were used for Fig. 1A-D      (A, B) Immunofluorescence images (A) and quantification of the density of cilia (B) in LX-2 cells treated with TGF-β for 24 h (n = 6 independent experiments).To quantify the percentage of ciliated cells (B), >140 cells were analyzed for each experiment.Scale bar, 10 µm.(C) IFT88 mRNA expression was measured by quantitative RT-PCR after TGF-β treatment for 24 h in LX-2 cells (n = 3 independent experiments).(D-F) Immunoblotting (D) of IFT88 and α-SMA in LX-2 cells treated with TGF-β, and the levels of IFT88 (E) and α-SMA (F) were quantified by densitometry (n = 3 independent experiments).(G, H) The effect of TGF-β on the half-life of IFT88 in LX-2 cells treated with CHX (20 mg/mL) was examined by immunoblotting (G), and the protein half-life curves were obtained (H) (n = 3 independent experiments).(I, J) LX-2 cells were treated with TGF-β for 24 h and then treated with MG132 (5 mM) for 12 h.The levels of IFT88 and GAPDH were examined by immunoblotting (I), and the level of IFT88 was determined by densitometry (J) (n = 3 independent experiments).Data information: Data are presented as mean ± SD.Statistical significance was determined by one-way ANOVA with post hoc tests (E, F) or unpaired two-tailed Student's t test (B, C, H, J). ns not significant; *P < 0.05, **P < 0.01, ***P < 0.001.Related to Fig. 3. Source data are available online for this figure.

Figure EV2 .
Figure EV2.Persistent presence of primary cilia on BECs and PVCs during CCl 4 -induced liver fibrosis.
and Fig. EV2.Data information: Data are presented as mean ± SD.Statistical significance was determined by one-way ANOVA.ns not significant.Related to Fig. 1.Source data are available online for this figure.

(
A-C) Immunofluorescence images (A) and quantification of the levels of α-SMA (B) and vimentin (C) in the liver of Ift88 fl/fl and Ift88 fl/fl ;Ubc-CreER mice treated with CCl 4 or corn oil (vehicle) for 2 months (n = 6 mice).Nuclei were stained with DAPI (blue).Scale bar, 50 μm.(D, E) CCl 4 -induced liver fibrosis in Ift88 fl/fl and Ift88 fl/fl ;Ubc-CreER mice was examined with Sirius red staining and H&E staining (D), and the percentage of collagen-positive areas was quantified (E) (n = 6 mice).Scale bars for Sirius red staining and H&E staining, 200 μm.Scale bar for liver, 1 cm.(F, G) Examination of the activities of AST (F) and ALT (G) in the serum of Ift88 fl/fl and Ift88 fl/fl ;Ubc-CreER mice (n = 4 mice).(H-L) The mRNA levels of IL-6 (H), IFNAR2 (I), TNF-α (J), COL1A1 (K), and α-SMA (L) in liver tissues were measured by quantitative RT-PCR (n = 4 mice).(M, N) Immunoblotting (M) and quantification (N) of the levels of IFT88 and α-SMA in the liver of Ift88 fl/fl and Ift88 fl/fl ;Ubc-CreER mice treated with CCl 4 or vehicle (n = 4 mice).Data information: Data are presented as mean ± SD.Statistical significance was determined by two-way ANOVA with post hoc tests.ns not significant; *P < 0.05, **P < 0.01, ***P < 0.001.Related to Fig. 2. Source data are available online for this figure.

Figure EV4 .
Figure EV4.Whole-body deficiency in IFT88 exacerbates CCl 4 -induced liver fibrosis over a 4-month period.(A-C) Immunofluorescence images (A) and quantification of α-SMA (B) and vimentin (C) in the liver of Ift88 fl/fl and Ift88 fl/fl ;Ubc-CreER mice treated with CCl 4 or corn oil (vehicle) for 4 months (n = 6 mice).Scale bar, 50 μm.(D, E) CCl 4 -induced liver fibrosis in Ift88 fl/fl and Ift88 fl/fl ;Ubc-CreER mice for 4 months was evaluated with Sirius red staining and H&E staining (D).The Image J software was used for the quantification of collagen-positive areas (E) (n = 6 mice).Scale bars for Sirius red staining and H&E staining, 200 μm.Scale bar for liver, 1 cm.(F, G) The activities of AST (F) and ALT (G) in the serum were analyzed in Ift88 fl/fl and Ift88 fl/fl ;Ubc-CreER mice (n = 4 mice).(H-L) The mRNA levels of IL-6 (H), IFNAR2 (I), TNF-α (J), COL1A1 (K), and α-SMA (L) in liver tissues were measured by quantitative RT-PCR (n = 4 mice).Data information: Data are presented as mean ± SD.Statistical significance was determined by two-way ANOVA with post hoc tests.ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001.Related to Fig. 2. Source data are available online for this figure.